The development of methods for expanding hematopoietic stem cells (HSC) in culture is under intense investigation and could provide unique opportunities both for clinical bone marrow transplantation (BMT) and for several gene therapy approaches, including the induction of tolerance to foreign transplantation antigens. While improvement of culture methods should provide a means both for increasing the efficiency of gene transfer into HSC and for selecting and expanding these cells once they are transduced, preclinical animal models are also essential in order to permit functional testing of transduced and cultured HSC in vivo. Although studies in mice have proven useful in this regard, large animal models are needed in which the ontogeny and transplantation biology of HSC are more closely related to those of humans. The miniature swine model has several advantages over other large animal models for this purpose, including defined transplantation genetics and the availability of monoclonal antibody and recombinant cytokine reagents. The overall objective of this research proposal is therefore to derive conditions which lead to the effective culture and retrovirus-mediated transduction of HSC from miniature swine, and to test these cells for their repopulating ability in vivo. During the preceding grant period we developed many of the basic techniques needed for these studies, including progenitor colony assays and long-term bone marrow culture systems. In addition, we used these techniques along with our established BMT model to demonstrate the reproducible transduction of swine HSC with retrovirus expression vectors for allogeneic MHC genes, as evidenced by the persistence of vector expression in multiple lineages in vivo for over one year. We will now extend these studies, focusing both on the improvement of HSC culture and transduction methods and on the development of in vivo reconstitution assays. In order to allow comparisons of different approaches in a single animal, two competitive repopulation assays will be developed: one involving allogeneic BMT with marrow from two separate, MHC-matched donors expressing different alleles of a common leukocyte cell surface antigen; and the other involving autologous BMT with separate aliquots of marrow transduced with genetically distinct retrovirus vectors. Several methods for improving the rate of retrovirus-mediated transduction and ex vivo expansion of swine HSC will be evaluated, including the use of recombinant swine hematopoietic cytokines, the support of HSC in culture with bone marrow stromal cell lines, the enrichment of HSC prior to transduction and culture, and the use of HSC from umbilical cord blood. In addition, culture methods will be optimized in order to increase the period of HSC exposure to virus and to select HSC following transduction. Finally, alternative methods for achieving engraftment with transduced and expanded HSC without lethal myeloablation will be tested.
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